Biomaterials (v.27, #14)

Calendar (I).

Well-defined poly((2-dimethyl amino)ethyl methacrylate-co-2-hydroxyethyl methacrylate)-b-poly(N-isopropylacrylamide)-b-poly((2-dimethyl amino)ethyl methacrylate-co-2-hydroxyethyl methacrylate), or P(DMAEMA-co-HEMA)-b-P(NIPAAm)-b-P(DMAEMA-co-HEMA), triblock copolymers were synthesized by consecutive atom transfer radical polymerizations (ATRPs), using ethylene glycol di-2-bromoisobutyrate (Br–EG–Br) as the starting ATRP initiator. The hydroxyl groups of the incorporated HEMA units were used as crosslinking sites for the preparation of smart hydrogels. The so-prepared hydrogels exhibited both temperature- and pH-sensitive behavior derived, respectively, and independently, from the P(NIPAAm) blocks and P(DMAEMA) units, in the crosslinked matrices. The hydrogels exhibited a lower critical solution temperature (LCST) of 31–32 °C in aqueous media of pH 1–7, not unlike that of the P(NIPAAm) homopolymer. The swelling ratios and swelling/deswelling kinetics of the hydrogels depended strongly on pH and temperature of the medium. The copolymers were characterized by gel-permeation chromatography, X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared (FTIR) spectroscopy, and 1H nuclear magnetic resonance (1H NMR) spectroscopy. The resultant stimuli-responsive hydrogels were characterized by differential scanning calorimetry (DSC). These stimuli-responsive hydrogels will have potential applications in biomedical areas, such as tissue engineering and drug delivery.
Keywords: Temperature-sensitive; pH-responsive; Hydrogel; NIPAAm; DMAEMA; ATRP;

Better materials are needed to promote bone growth. For this reason, the present study created nanometer crystalline hydroxyapatite (HA) and amorphous calcium phosphate compacts functionalized with the arginine-glycine-aspartic acid (RGD) peptide sequence. Crystalline HA and amorphous calcium phosphate nanoparticles were synthesized by a wet chemical process followed by a hydrothermal treatment for 2 h at 200 °C and 70 °C, respectively. Resulting particles were then pressed into compacts. For the preparation of conventional HA particles (or those with micron diameters), the aforementioned pressed compacts were sintered at 1100 °C for 2 h. Peptide functionalization was conducted by means of a three step reaction procedure: silanization with 3-aminopropyltriethoxysilane (APTES), cross-linking with N-succinimidyl-3-maleimido propionate (SMP), and finally peptide immobilization. The three step reaction procedure was characterized by a novel 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) fluorescence technique. For all materials, results showed that the immobilization of the cell adhesive RGD sequence increased osteoblast (bone-forming cell) adhesion compared to those non-functionalized and those functionalized with the noncell adhesive control peptide (RGE) after 4 h. However, surprisingly, results also showed that the adhesion of osteoblasts on non-functionalized amorphous nanoparticulate calcium phosphate was similar to conventional HA functionalized with RGD. Osteoblast adhesion on nanocrystalline HA (unfunctionalized and functionalized with RGD) was below that of the respective functionalized amorphous calcium phosphate but above that of the respective functionalized conventional HA. In this manner, results of this study suggest that decreasing the particulate size into the nanometer regime and reducing crystallinity of calcium phosphate based materials may promote osteoblast adhesion to the same degree as the well-established techniques of functionalizing conventional HA with RGD.
Keywords: Nanomaterials; Hydroxyapatite; Osteoblast adhesion; RGD grafting; Orthopedic; Nanotechnology;

Examination of the effects of poly(N-vinylpyrrolidinone) hydrogels in direct and indirect contact with cells by Louise Elizabeth Smith; Stephen Rimmer; Sheila MacNeil (2806-2812).
Poly(N-vinylpyrrolidinone) (PNVP) has been used in various biomedical applications for many years. This study explores two PNVP hydrogels for their biocompatibility with skin cells and their ability to support the growth of skin cells in direct and indirect contact with the cells. Two crosslinked PNVP's were investigated, one crosslinked with ethylene glycol dimethacrylate (EGDMA) and the other crosslinked with diethylene glycol bisallylcarbonate (DEGBAC). The different crosslinkers lead to hydrogels with different mechanical and slightly different biological properties. While neither hydrogel proved to be a suitable substrate for culturing cells (based on fibroblasts and a range of other cells), indirect contact with both showed them to be biocompatible and even stimulatory to fibroblasts. The P(NVP-co-DEGBAC) hydrogel stimulated fibroblast viability more reliably than the P(NVP-co-EGDMA) hydrogel when in indirect contact with cells. This effect was shown to be independent of the presence of foetal calf serum in the culture media, and could not be explained by any hydrogel breakdown products during the course of these experiments. Rather the phenomenon was observed to be the result of a dynamic interaction between the hydrogels and the cells.
Keywords: Cell viability; Copolymer; Fibroblast; Hydrogel; Poly(N-vinylpyrrolidinone);

Cardiovascular implants such as vascular grafts fail frequently because they lack genuine blood-compatibility. The blood-contacting surface should simultaneously prevent thrombus formation and promote formation of a confluent endothelial cell layer, to achieve sustained haemostasis. Contact activation and endothelialization are known to be determined by the plasma proteins which adsorb onto virtually all synthetic surfaces almost immediately upon contact with blood. A common approach in blood-compatibility research is, therefore, to use hydrophilic biomaterials, which are sometimes claimed to be “protein-repellent”. We report here that, for synthetic polymeric surfaces, hydrophilicity is by no means synonymous to protein-repellency. We discovered that significant amounts of proteins, especially high-density lipoprotein, adsorb to hydrophilic surfaces. Pre-incubation of hydrophilic synthetic surfaces with high-density lipoprotein provides a blood–biomaterial interface, which inhibits thrombin generation and subsequent thrombus formation, and also accommodates overgrowth with a confluent endothelial layer. This approach may open the way to truly functional small-caliber arterial prostheses, and may also be relevant to cardiovascular tissue engineering in which de novo vascular tissues are cultured on or within a biomaterial scaffold.
Keywords: Endothelialization; Protein adsorption; Vascular graft; Hydrophilicity; Blood compatibility;

Uptake of functionalized, fluorescent-labeled polymeric particles in different cell lines and stem cells by Myriam Ricarda Lorenz; Verena Holzapfel; Anna Musyanovych; Karin Nothelfer; Paul Walther; Hendrik Frank; Katharina Landfester; Hubert Schrezenmeier; Volker Mailänder (2820-2828).
Labeling of cells with particles for in-vivo detection is interesting for various biomedical applications. The objective of this study was to evaluate the feasibility and efficiency labeling of cells with polymeric particles without the use of transfection agents. We hypothesized that surface charge would influence cellular uptake. The submicron particles were synthesized by the miniemulsion process. A fluorescent dye which served as reporter was embedded in these particles. The surface charge was varied by adjusting the amount of copolymerized monomer with amino group thus enabling to study the cellular uptake in correlation to the surface charge. Fluorescent-activated cell sorter (FACS) measurements were performed for detecting the uptake of the particles or attachment of particles in mesenchymal stem cells (MSC), and the three cell lines HeLa, Jurkat, and KG1a. These cell lines were chosen as they can serve as models for clinically interesting cellular targets. For these cell lines—with the exception of MSCs—a clear correlation of surface charge and fluorescence intensity could be shown. For an efficient uptake of the submicron particles, no transfection agents were needed. Confocal laser scanning microscopy and transmission electron microscopy (TEM) revealed differences in subcellular localization of the particles. In MSCs and HeLa particles were mostly located inside of cellular compartments resembling endosomes, while in Jurkat and KG1a, nanoparticles were predominantly located in clusters on the cell surface. Scanning electron microscopy showed microvilli to be involved in this process.
Keywords: Particle; Fluorescence; Cell uptake; Surface modification; Mesenchymal stem cell; FACS; SEM; TEM; Confocal microscopy;

Quantitative kinetic analysis of gene expression during human osteoblastic adhesion on orthopaedic materials by Myriam Rouahi; Eric Champion; Pierre Hardouin; Karine Anselme (2829-2844).
Little information was found in the literature about the expression on hydroxyapatite (HA) materials of genes specific of cellular adhesion molecules although more were found on titanium-based substrates. Hence, the goal of this work was to study by a kinetic approach from 30 min to 4 days the adhesion of Saos-2 cells on microporous (mHA) and non-microporous hydroxyapatite (pHA) in comparison to polished titanium. Our strategy associated the visualization of adhesion proteins inside the cells by immunohistochemistry and the quantitative expression of genes at mRNA level by real-time PCR. The cell morphology was assessed using scanning electron microscopy and the number of cells thanks to biochemical techniques. The cellular attachment was the highest on mHA from 30 min to 24 h although the cell growth on mHA was the lowest after 4 days. Generally, the Saos-2 osteoblastic cells morphology on mHA was radically different than on other surfaces with the particularity of the cytoplasmic edge, which appeared un-distinguishable from the surface. The revelation by specific antibodies of proteins of the cytoskeleton (actin) and the focal adhesions (FAK, phosphotyrosine) confirmed that adhesion and spreading were different on the 3 materials. The actin stress fibres were less numerous and shorter on mHA ceramics. Cells had more focal contacts after 4 h on mHA compared to other substrates but less after 24 h. The highest values of total proteins were extracted from mHA at 0.5 and 24 h and from pHA at 1, 4, and 96 h. The αv and β1 integrin, actin, FAK, and ERK gene expression were found to be different with adhesion time and with materials. C-jun expression was comparable on mHA, titanium and plastic but was largely higher than on pHA at 0.5 and 1 h. On the contrary, c-fos expression was the highest on pHA after 0.5 h and the lowest after 1 h. This difference between c-fos and c-jun expression on pHA after 0.5 h could be related to the fact that these two genes may differ in their signalling pathways. The expression of the alkaline phosphatase gene after 4 days was lower on mHA compared to other materials demonstrating that the microstructure of the mHA ceramic was not favourable to Saos-2 cells differentiation.Finally, it was demonstrated in this study that HA and titanium surfaces influence as well gene expression at early times of adhesion as the synthesis of adhesion proteins but also proliferation and differentiation phases. Indeed, the signal transduction pathways involved in adhesion of Saos-2 cells on HA and titanium were confirmed by the sequential expression of αv and β1 integrins, FAK, and ERK genes followed by the expression of c-jun and c-fos genes for proliferation and alkaline phosphatase gene for differentiation.
Keywords: Hydroxyapatite; Osteoblasts; Cell adhesion; Real-time PCR; Immunochemistry; Gene expression;

In vitro spermatogenesis by three-dimensional culture of rat testicular cells in collagen gel matrix by Jae Ho Lee; Hyun Joo Kim; Haekwon Kim; Sang Jin Lee; Myung Chan Gye (2845-2853).
In an effort to improve in vitro spermatogenesis by potentiating the interactions between developing germ cells, somatic cells, and the extracellular matrix (ECM), the efficiency of the germ cell-somatic cell coculture in a three-dimensional (3D) collagen gel matrix was examined. Cells isolated from rat seminiferous tubules 18 days after birth were cultured for 22 days in a monolayer without ECM, collagen gel (CG), or collagen+Matrigel (CGM). After culture, the viabilities of the cultured cells in the monolayer, CG, and CGM culture were 42.8%, 70.7% and 76.1%, respectively. Occludin-positive cells in a cyst-like structure were found in the ECM gel matrix together with 3β hydroxysteroid dehydrogenase-positive cells, suggesting the presence of functional Sertoli cells and Leydig cells, respectively. Flow cytometric analysis of DNA content revealed a significant increase in the haploid cell population in the CG and CGM compared to the monolayer culture. Transition protein 2 (TP2) and protamine 2-positive cells were found together with a significant increase in TP2 mRNA levels in cells cultured in CG and CGM over those in monolayer culture, suggesting the occurrence of the post-meiotic differentiation of spermatogenetic cells. Taken together, a 3D in vitro culture system for testicular cells using a collagen gel matrix could enhance viability, meiosis, and post-meiotic differentiation of germ cells to presumptive differentiating spermatids.
Keywords: Spermatogenesis; Three-dimensional culture; Collagen;

The influence of architecture on degradation and tissue ingrowth into three-dimensional poly(lactic-co-glycolic acid) scaffolds in vitro and in vivo by Yang Cao; Geraldine Mitchell; Aurora Messina; Lisa Price; Erik Thompson; Anthony Penington; Wayne Morrison; Andrea O’Connor; Geoffrey Stevens; Justin Cooper-White (2854-2864).
The in vitro and in vivo degradation properties of poly(lactic-co-glycolic acid) (PLGA) scaffolds produced by two different technologies—thermally induced phase separation (TIPS), and solvent casting and particulate leaching (SCPL) were compared. Over 6 weeks, in vitro degradation produced changes in SCPL scaffold dimension, mass, internal architecture and mechanical properties. TIPS scaffolds produced far less changes in these parameters providing significant advantages over SCPL. In vivo results were based on a microsurgically created arteriovenous (AV) loop sandwiched between two TIPS scaffolds placed in a polycarbonate chamber under rat groin skin. Histologically, a predominant foreign body giant cell response and reduced vascularity was evident in tissue ingrowth between 2 and 8 weeks in TIPS scaffolds. Tissue death occurred at 8 weeks in the smallest pores. Morphometric comparison of TIPS and SCPL scaffolds indicated slightly better tissue ingrowth but greater loss of scaffold structure in SCPL scaffolds. Although advantageous in vitro, large surface area:volume ratios and varying pore sizes in PLGA TIPS scaffolds mean that effective in vivo (AV loop) utilization will only be achieved if the foreign body response can be significantly reduced so as to allow successful vascularisation, and hence sustained tissue growth, in pores less than 300 μm.
Keywords: Polyglycolic acid and polylactic acid scaffold; In vitro and in vivo tests; Biodegradation; Mechanical properties; Foreign body response;

The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components by Lee Graham; Paul R. Cooper; Nicola Cassidy; Jacques E. Nor; Alastair J. Sloan; Anthony J. Smith (2865-2873).
Calcium hydroxide (Ca(OH)2) has been used extensively to induce dentine regeneration through formation of dentine bridges at sites of pulp exposure after dental tissue injury, however, the biological processes underpinning these events are unclear. We hypothesise that growth factors and other bio-active molecules, sequestered within dentine matrix, may be released by the action of Ca(OH)2 and signal gene expression in pulp cells, which mediates the changes in cell behaviour observed during regeneration. Powdered sound, human dentine samples were extracted with either 0.02  m Ca(OH)2, pH 11.7 or 10% EDTA, pH 7.2 ( a control known extractant of bio-active and other ECM molecules from dentine) over a 14-day period. Extracts were compared for non-collagenous protein (NCP) and glycosaminoglycan (GAG) content using dye binding assays and protein compositions were analysed by 1D-polyacrylamide gel electrophoresis (1D-PAGE) and TGF-β1 ELISA. The effects of extracts on TGF-β1, Collagen-1α and Nestin gene expression were analysed using semi-quantitative RT-PCR in the dental MDPC-23, OD-21 and fibroblastic Swiss 3T3 cell lines following 24 h of exposure. Ca(OH)2 solubilised NCPs and GAGs from the dentine ECM, although with a lower yield than the EDTA solution and with different kinetics. 1D-PAGE analysis demonstrated some differences in profiles for proteins solubilised from dentine by Ca(OH)2 and EDTA. Both solutions released TGF-β1 from the dentine with higher concentrations present in the EDTA (1.395±0.036 ng/mg) versus the Ca(OH)2 (0.364±0.012 ng/mg) extract. Notably, both extracts induced similar gene expression profiles in all cell lines. These data provide a rational explanation for the action of Ca(OH)2 during pulp capping in which the cellular activities involved in dentine bridge formation may be mediated through release of growth factors and other bio-active molecules from the dentine by Ca(OH)2.
Keywords: Calcium hydroxide; Dentine; Pulp; Regeneration; Growth factors; TGF;

Bone marrow cell gene expression and tissue construct assembly using octacalcium phosphate microscaffolds by R.M. Shelton; Y. Liu; P.R. Cooper; U. Gbureck; M.J. German; J.E. Barralet (2874-2881).
Calcium phosphates have been widely used in bone and soft tissue applications and are of considerable interest as scaffold materials due to properties of osteoconduction, resorbability and in some cases osteoinduction. These materials are microcrystalline and as such are processed using sintering, surface coating or cement technologies. However calcium phosphates containing HPO4 2− ions often have layered crystal structures and can form macrocrystals in an aqueous environment at room temperature and pressure. This study aimed to investigate the potential of octacalcium phosphate (OCP) crystals for the attachment, proliferation and differentiation of bone marrow stromal cells and the potential of these cell seeded crystals as ‘building blocks’ for manufacture of self-supporting macroscale tissue constructs. An inverse relationship between cell number and crystal surface area was found and marrow cells grown on OCP crystals expressed osteocalcin and osteopontin mRNA, markers of osteoblastic differentiation, even in the absence of inductive media additives. Self-supporting crystal tissue macroscale constructs could be fabricated by culturing cell loaded crystals in moulds of the desired shape. Due to the low packing efficiency as a consequence of the high aspect ratio of OCP crystals, this microscaffold approach may offer the potential for ex vivo construction of large volumes of tissue which forms as a physiologically vascularised tissue.
Keywords: Bone graft; Bone tissue engineering; Calcium phosphate; Scaffold; Osteoblast; Octacalcium;

Treatment of articular cartilage defects in horses with polymer-based cartilage tissue engineering grafts by Dirk Barnewitz; Michaela Endres; Ina Krüger; Anja Becker; Jürgen Zimmermann; Ingo Wilke; Jochen Ringe; Michael Sittinger; Christian Kaps (2882-2889).
The objective of our study was to evaluate the integration of autologous cartilage tissue engineering transplants based on resorbable polyglactin/polydioxanone scaffolds into full-thickness cartilage defects of horses. Cartilage biopsies were taken from the non-load-bearing area of the lateral talus of the left tibiotarsal joint of eight healthy Haflinger horses. Tissue engineering cartilage transplants were generated by three-dimensional arrangement of autologous chondrocytes in biocompatible and resorbable polymer scaffolds. Full-thickness cartilage defects of 8 mm in diameter were created in the tubular bone condyle of the fetlock joint and cartilage grafts were fixed using an anchor system, while defects without grafting served as controls. After 6 and 12 months the repair tissue was evaluated histologically and showed formation of a cartilaginous tissue and good integration into the surrounding host tissue with firm bonding of the graft to the adjacent cartilage and the underlying subchondral bone. Biochemical analysis demonstrated that the content of glycosaminoglycans and hydroxyproline is comparable in repair tissue derived from treated and control defects. The use of three-dimensional autologous cartilage transplants based on resorbable polymer scaffolds ensures secure fixation, good integration of the graft into cartilage lesions, and is therefore suggested as a promising therapeutic option for the treatment of cartilage defects.
Keywords: Tissue engineering; Autologous cartilage repair; Cartilage regeneration; Polyglactin/polydioxanone scaffold; Horse model;

Control of cell adhesion on poly(methyl methacrylate) by Shyam Patel; Rahul G. Thakar; Josh Wong; Stephen D. McLeod; Song Li (2890-2897).
Keratoprostheses have been constructed from a wide variety of transparent materials, including poly(methyl methacrylate) (PMMA). However, the success of keratoprosthesis has been plagued by numerous shortcomings that include the weakening of the implant-host interface due to weak cell adhesion and opaque fibrous membrane formation over the inner surface of the implant due to fibroblast attachment. An effective solution requires a surface modification that would selectively allow enhanced cell attachment at the implant-host interface and reduced cell attachment over the interior surface of the implant. Here, we have developed a novel and simple peptide conjugation scheme to modify PMMA surfaces, which allowed for region-specific control of cell adhesion. This method uses di-amino-PEG, which can be grafted onto PMMA using hydrolysis or aminolysis method. PEG can resist cell adhesion and protein adsorption. The functionalization of grafted di-amino-PEG molecules with RGD peptide not only restored cell adhesion to the surfaces, but also enhanced cell attachment and spreading as compared to untreated PMMA surfaces. Long-term cell migration and micropatterning studies clearly indicated that PEG–PMMA surfaces with and without RGD conjugation can be used to differentiate cell adhesion and control cell attachment spatially on PMMA, which will have potential applications in the modification of keratoprostheses.
Keywords: Poly(methylmethacrylate) (PMMA); Keratoprosthesis; Surface modification; Cell adhesion; Antiadhesion; Micropatterning;

Double-walled microspheres (DWMS), with drug localized to the particle core, present a promising route for control of drug release rates, for example, by varying the degradation rate or erosion mechanism of the polymer used to form the shell or the thickness of the shell. DWMS are often difficult to fabricate, however. Thermodynamic descriptions for polymer–polymer immiscibility, drug distribution between phases and polymer–solution spreading coefficient provide predictions of appropriate solvents and polymer concentrations for efficiently producing well-formed DWMS. As an example, thermodynamic parameters for a polyphosphoester/poly(d,l-lactide-co-glycolide) (PLG) DWMS system, encapsulating piroxicam, have been calculated and the predictions tested experimentally. Appropriate choices of solvents and initial polymer concentrations resulted in DWMS with the desired polyphosphoester shells and piroxicam located selectively in PLG cores.
Keywords: Microcapsules; Double-walled microspheres; Microencapsulation; Controlled drug release; Piroxicam;